Method for the detection of malignant diseases

ABSTRACT

In order to aid in the detection of malignant diseases the sample of a body fluid is incubated with at least two receptors R 1  and R 2  in which a signal change is produced by binding of at least the receptors R 1  and R 2  to the substance to be detected in the sample solution and in which one of the two receptors contains a monoclonal antibody which binds to the amino acid sequence 311 to 335 of cytokeratin 19 and the other receptor contains a monoclonal antibody which binds to the amino acid sequence 346 to 359 of cytokeratin 19 and the signal change in the sample caused by the binding is determined.

The invention concerns a method for the detection of malignant diseases and a suitable reagent therefor.

In clinical diagnostics the search for an indicator substance which can indicate malignant diseases has already been going on for a long time. Hopes have been placed on CEA (carcinoembryonic antigen) and TPA (tissue polypeptide antigen) as tumour markers with a broad organ specificity. Meanwhile, however, it has turned out that both proteins could not fulfill these expectations. The application of CEA has in the meantime been reduced mainly to therapeutic monitoring e.g. of colorectal carcinomas while TPA has only gained acceptance for a few indications in therapeutic monitoring because of its lack of specificity and sensitivity. Very many other proteins have been tested as indicators for malignant diseases but they were all unsatisfactory.

The cytokeratin (CK) class has also been associated with tumours. In the form of intermediary filament proteins they are components of the cytoskeleton of epithelial cells. Nineteen cytokeratins are known of which the cytokeratins 1 to 8 are denoted basic and the cytokeratins 9 to 19 acidic cytokeratins. The cytokeratins can aggregate in the cell to form tetramers. Each tetramer consists of two basic and two acidic cytokeratin molecules. Filaments are produced by the linear aggregation of tetramers. Intact cytokeratin molecules are water-insoluble as integral components of the intermediary filaments of epithelial cells. The complexity and composition of cytokeratins is different in the various epithelial tissues i.e. epithelial cells have cytokeratin compositions which are typical for the tissue. However, up to now nothing has been known about whether malignant diseases correlate with the occurrence of cytokeratins in body fluids.

It was thus the object of the present invention to provide a method with which the presence of a malignant disease can be diagnosed.

This object is achieved by a method for the detection of malignant diseases which is characterized in that the sample of a body fluid is incubated with at least two receptors R₁ and R₂ in which a signal change is produced by binding of at least one of the receptors R₁ and R₂ to the substance to be detected in the sample solution and in which one of the two receptors contains a monoclonal antibody which binds to the amino acid sequence 291 to 335 of cytokeratin 19 and the other receptor contains a monoclonal antibody which binds to the amino acid sequence 346 to 367 of cytokeratin 19 and the signal change in the sample caused by the binding is determined.

Surprisingly it was established that fragments of cytokeratin 19 are formed primarily in epithelial tumour tissues which have at most the amino acid sequence 219 to 367, but at least the sequence 311 to 359, of the complete cytokeratin 19 and have epitopes on the amino acid sequences 291 to 335 and 346 to 367 which can specifically bind to the aforementioned monoclonal antibodies or to derivatives thereof and which, in addition, can be found in body fluids. Cytokeratin 19 (CK 19) has an amino acid chain consisting of 400 amino acids. The sequence is described in Stasiak, P. C. et al., J.Invest.Dermatol. 92 (1989) 707-716, in particular on page 712 and is divided into 3 domains 1A, 1B and 2. The fragments which are to be detected according to the present invention originate from domain 2 (helix 2). It was found that these CK 19 fragments which bind specifically to both of the above-mentioned antibodies are detectable in particular in bronchial, breast, stomach, biliary tract, liver and colon carcinomas. These fragments could not as a rule be detected in patients with inflammatory epithelial diseases of the respective tissues.

The monoclonal antibodies which are formed by the cell lines ECACC 89112803 (binds to the sequence 291 to 335, preferably to the sequence 311 to 335) and ECACC 89112804 (binds to the amino acid sequence 346 to 367, preferably to 346 to 359) are preferably used for the invention.

Other monoclonal antibodies (mAB) which bind to CK 19 but which do not bind to the CK 19 sequences defined in claim 1 are not at all suitable for the method according to the present invention or result in a reaction with insufficient clinical specificity. This is for example demonstrated by comparison studies according to Example 3 which were carried out with the mABs AE1 and b170. AE1 binds to an epitope on the amino acid sequence 153 to 219 and is described in J.Biol.Chem. 261 (1986) 4646-4654 and in J.Cell.Biol 95 (1982) 580-588. b170 binds to an epitope on the amino acid sequence 336-345, hence between the two preferred mABs of the invention and is obtainable according to Lab.Invest. 55 (1986) 497-504. The results of these experiments are shown in the following Table:

    ______________________________________                                                  malignant diseases                                                                          benign diseases                                                     examined           examined                                         mAB combination                                                                           sera      positive sera    positive                                 ______________________________________                                         803.sup.1) + 804.sup.2)                                                                   24        11       24      4                                        803 + b170 24        0        24      2                                        b170 + 804 24        4        24      1                                        AE1 + 804  24        0        24      0                                        ______________________________________                                          .sup.1) = ECACC 89112803                                                       .sup.2) = ECACC 89112804                                                 

The test is carried out in body fluids, preferably serum. The determination is carried out according to generally known immunological methods. Very many variants are known for carrying out the intended immunological method of determination according to the present invention, all of which are suitable in this case. Thus, two or three or even more receptors can be used and incubation with the individual receptors can be carried out in different sequences in a homogeneous or heterogeneous phase. In each case the change in signal which results from the binding of at least two receptors to the fragment to be detected in the sample solution is evaluated. The variants of the methods are known to the expert and do not need to be elucidated here in more detail. The determination according to the present invention is preferably carried out either in a homogeneous phase e.g. based on an agglutination assay in which coated particles are used as receptors such as e.g. latex particles or erythrocytes which cross-link and thereby agglutinate by binding to the specifically bindable receptors and the substance to be determined, or in a heterogeneous phase, preferably as a sandwich immunoassay. In every case at least two receptors R₁ and R₂ are used of which one contains a monoclonal antibody which binds to the sequence 291 to 335 of CK 19, while the other receptor contains a monoclonal antibody which binds to the sequence 346 to 367 of CK 19. Antibodies which have an adequate epitope overlap with the antibody under consideration are also suitable. This epitope overlap can be easily detected with the aid of a competitive test system. For this purpose the extent to which an antibody competes with an antibody, which was obtained by using one or both of the aforementioned binding sequences as immunogen, for binding to a defined substrate or to a special epitope is investigated e.g. with the aid of an enzyme-immunoassay. For this a solution containing the fragment of the corresponding sequence is incubated with the defined monoclonal antibody produced according to the invention in labelled form and with an excess of the antibody under consideration. By immobilization of the complexes formed, separation of the solid from the liquid phase and detection of the bound label in one of the two phases, it is then easy to establish to what extent the monoclonal antibody under consideration can displace the defined antibody from the binding. If the displacement is at least 50% at a 10⁵ -fold excess, then an epitope overlap is present and the corresponding antibody is suitable for use in the method according to the present invention.

mABs which are suitable for the invention are obtained by methods known for this purpose to one skilled in the art using CK 19 fragments which contain suitable sequences defined above or consist thereof. Complete CK 19 can also be used.

In the incubation of the body fluid with the two receptors, complexes form from R₁, cytokeratin 19 fragment and R₂. The receptors are selected such that only complexes in which R₁ as well as R₂ are bound to the cytokeratin 19 fragment generate a change in signal and thus in this way only those fragments are detected which are capable of binding to both specific antibodies.

The determination according to the present invention is preferably carried out as a sandwich immunoassay. For this receptor R₁ is immobilized or made to be immobilizable and reacted with the sample solution. Subsequently receptor R₂ is added. Complexes form from the immobilized receptor R₁, the CK 19 fragment to be detected and receptor R₂. Only complexes which are bound to the solid phase and carry a label enter into the evaluation.

In this embodiment receptor R₁ mediates the binding to the solid phase. For this receptor R₁ can either be bound directly to the solid phase or via a spacer or it can even be immobilizable. In a preferred embodiment receptor R₁ is a conjugate of a monoclonal antibody having the above-mentioned specificity and a specifically bindable substance. The partner capable of binding to the specifically bindable substance is bound to a solid phase. As specifically bindable pairs mention may be made of for example antigen-antibody, hapten-antibody, biotin-antibiotin-antibody, biotin-avidin, biotin-streptavidin, protein-A-immuno-γ-globulin. In this embodiment it is particularly preferably to use as R₁ a conjugate of the above-mentioned monoclonal antibody with biotin and a matrix which carries streptavidin on its surface as the solid phase. The immobilization of the monoclonal antibody is then effected by binding of the biotin to streptavidin. An embodiment is also preferred in which antibodies against the Fc part of the monoclonal antibody used for receptor R₁ or protein A molecules are bound to the surface of the solid phase whereby immobilization is then effected by binding of the Fc parts of the monoclonal antibodies of R₁.

In a further preferred embodiment biotin molecules are bound to a matrix and as receptor R₁ a conjugate of biotin and the monoclonal antibody is used. The immobilization can then be effected after carrying out the immunological reaction by addition of streptavidin.

The materials usually used in immunological methods are suitable as the solid phase. For example polymer materials as well as glass can be employed. Polystyrene, polymethacrylate, teflon, polyamide, copolymers of styrene and acrylonitrile, glass and cellulose products have proven to be especially suitable. The matrix can be present in any form e.g. as tubes, microtitre plates, beads, film, powder, particles or fibre pads. For example solid phases produced according to one of the processes described in DE-A 36 40 412 are suitable.

In this embodiment receptor R₂ contains in each case the other monoclonal antibody which is necessary according to the present invention. Receptor R₂ is labelled according to known methods. Radioactive substances, substances generating NMR signals, enzymes and fluorescent substances are suitable as the label. The detection of the label is carried out according to known methods. An enzyme is preferably employed as the label. Peroxidase, alkaline phosphatase and β-galactosidase are particularly suitable as enzymes. The enzyme is detected by addition of a substrate and measurement of the colour formed.

In the further preferred embodiment of the agglutination assay receptor R₁ is a particle coated with one of the two defined antibodies while the other receptor is a particle coated with the other antibody. Binding of the particle to the substance to be detected results in agglutination which can be detected by the change in turbidity.

The presence of particular fragments of cytokeratin 19 which contain two epitopes and are capable of binding to both antibodies employed can be detected with the method according to the present invention. Such fragments are formed primarily in tumour tissue.

The invention also provides a reagent for the detection of malignant diseases which is characterized in that it contains at least two receptors R₁ and R₂ in which one of the two receptors contains a monoclonal antibody which binds to the sequence 291 to 335 of cytokeratin 19 and the other receptor contains a monoclonal antibody which binds to the sequence 346 to 367 of cytokeratin 19 or an antibody capable of binding in an equivalent manner or its derivatives. The reagent preferably contains the mAB produced by the cell line ECACC 89112803 and that produced by the cell line ECACC 89112804.

The invention also provides the cell cultures ECACC 89112804 and ECACC 89112803 deposited at the European Collection of Animal Cell Cultures, Porton Down (GB) which produce antibodies against cytokeratin 19 and the antibodies themselves produced by these cell lines.

Because of their reactivity with the aforementioned epitopes of cytokeratin 19 each of the two antibodies can be used to differentiate in vivo between epitope positive and epitope negative tissues. For this the antibody or its Fab or (Fab')₂ fragments are bound to a label which is suitable for this purpose and conveyed to epitope positive tissue (e.g. tumours, metastases) via suitable transport media e.g. via the blood stream after injection and are bound there. The bound antibody can then be visualized using imaging techniques. Examples of labels which can be visualized are the isotopes Tc-99, I-131, I-125, In-111 for radiographic imaging and Fe, Cu, Mn, Gd or F for imaging by nuclear magnetic resonance.

By this means a method is also made possible for the diagnosis of malignant diseases in vivo in which at least one of the two receptors, which are used according to the present invention, is conveyed to body tissue and its specific binding is determined by immunoscintigraphic means.

The invention is elucidated by the following Examples.

EXAMPLE 1

Monoclonal antibody BM 19 ECACC 89112804.

Immunogen

The cytoskeleton of MCF-7 cells serves as the immunogen. The principle used for the production of these immunogens is described inter alia in Meth. in Enzymol. 134, 355 ff. (1986) and Exp. Cell Res. 173, 17 ff (1987). In summary, an extract is made from ca. 10¹⁰ MCF-7 cells using detergent-buffer (1% Triton). After centrifuging the homogenates at 2500 g the residues were extracted with saline-buffer. The insoluble fraction remaining after this extraction corresponded to the CK fraction. This fraction was used as the immunogen.

Immunization

Balb/c mice, 6 to 8 weeks old, were immunized intraperitoneally with 70 μg of the CK fraction containing CK 19 antigen in complete Freund's adjuvant. 3 further immunizations each with 70 μg antigen in incomplete Freund's adjuvant were carried out at three-month intervals.

Fusion and cloning

Spleen cells of an immunized mouse were fused with X63-Ag8-653 myeloma cells (ATCC-CRL 8375) in a 1:1 ratio according to the standard procedure according to J. of Immunol. Meth., Vol. 39, 285-308.

During the subcloning the CK specific clones were selected out by their positive reaction in immunofluorescent microscopy. Culture cells (MCF-7) as well as human tissue (liver) were used for the immunofluorescent microscopy.

Induction and Ascites

2 to 5×10⁶ hybrid cells were injected intraperitoneally into mice which had been pretreated with Pristan. After 15 to 20 days ascites could be isolated with an antibody concentration of 5 to 10 mg/ml.

Specificity of the Monoclonal Antibody BM 19

The antibody has the subclass IgG2b. It reacts exclusively with CK 19 in blot analyses with CK from different tissues (e.g. epidermis, myometrium) and culture cells (e.g. MCF-7, RT 112, A 431).

EXAMPLE 2

Monoclonal antibody Ks 19.1 ECACC 89112803 (IgG2a)

Immunogen

Living cells of the human cell line MCF-7 served as immunogen.

Immunization

Balb/c mice were immunized intraperitoneally five times with ca. 10⁷ living cells.

Fusion and Cloning

An electrofusion was carried out (Eur. J. Clin. Oncol. 21, 733 ff (1985)). The plasmacytoma line X63-Ag8-653 served as the fusion partner.

Induction of Ascites

2 to 5×10⁶ hybrid cells were injected intraperitoneally into mice which had been pretreated with Pristan. After 10 to 15 days ascites could be isolated with an antibody concentration of 10 to 15 mg/ml.

EXAMPLE 3 Test Procedure for CK 19

A sandwich enzyme-immunoassay was carried out to determine the CK 19 fragment in body fluids. In this procedure the monoclonal antibody Ks 19.1 (3 μg/ml) was bound for one hour at room temperature as a biotin conjugate in a volume of 100 μl PBS to the streptavidin-coated well of a microtitre plate. After washing four times with 0.05% Tween/PBS the incubation with the serum sample (2 μl serum added to 100 μl PBS) was carried out for 90 minutes at room temperature. Afterwards it is washed again four times with 0.05% Tween/PBS. Subsequently it was incubated for 90 minutes at room temperature with the monoclonal antibody BM 19 coupled to peroxidase (final concentration 250 mU/ml). After again washing four times with 0.05% Tween/PBS it was incubated at room temperature with the enzyme substrate solution ABTS® (100 mmol/l phosphate-citrate buffer pH 5.0, 1.47 mmol/l sodium perborate, 9.1 mmol/l ABTS®) and after 30 minutes the absorbance at 405 nm was measured as a measure for the analyte concentration.

The tests were carried out in the sera of patients with various diseases. The results are shown in the following table.

                  TABLE                                                            ______________________________________                                         Test "Cytokeratin 19"                                                          Disease          sera examined                                                                              positive sera                                     ______________________________________                                         1.  normal sera      19          0                                             2.  benign diseases                                                                gravidae         20          2                                                 renal insufficiency                                                                             5           1                                                 pancreatitis     2           0                                                 cirrhosis of the liver                                                                          5           3                                                 Crohn's syndrome 5           0                                                 hepatitis        5           3                                                 mastopathy       1           0                                                 uterus myoma     2           0                                                 auto-immune disease                                                                             5           1                                                 prim. bil. cirrhosis                                                                            5           1                                             3.  malignant diseases                                                             breast carcinoma 37          20                                                ovarial carcinoma                                                                               8           3                                                 colon carcinoma  7           7                                                 pancreatic carcinoma                                                                            5           1                                                 gastric carcinoma                                                                               5           3                                                 bronchial carcinoma                                                                             9           4                                                 cervical carcinoma                                                                              5           3                                                 prim. liver cell ca.                                                                            5           4                                                 ENT carcinoma    3           0                                                 biliary carcinoma                                                                               4           4                                                 plasmacytoma     5           0                                                 metastasing carcinoma                                                                           2           0                                                 of the scrotum                                                                 carcinoma of the prostate                                                                       2           1                                                 neuroblastoma    1           0                                                 colitis ulc.     1           0                                             ______________________________________                                    

EXAMPLE 4

The overlap between the epitope of an antibody and the monoclonal antibody ECACC 89112804 was determined. The test was based on a competitive enzyme-immunoassay. For this the monoclonal antibody, e.g. ECACC 89112803 (3 μg/ml) was bound as a biotin conjugate for one hour at room temperature in a volume of 100 μl PBS (8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l NaH₂ PO₄ ×2H₂ O, 0.2 g/l KH₂ PO₄) to the streptavidin-coated well of a microtitre plate. After washing four times with 0.05% Tween 20/PBS it was incubated for 90 minutes at room temperature with a serum sample (2 μl serum added to 100 μl PBS) with a high titre. Afterwards it was washed again four times with 0.05% Tween 20/PBS. Subsequently it was incubated simultaneously for 90 minutes at room temperature with the monoclonal antibody, e.g. ECACC 89112804 labelled with peroxidase (final concentration 250 mU/ml) and the antibody to be evaluated. After washing again four times with 0.05% Tween 20/PBS it was incubated at room temperature with the enzyme substrate solution ABTS® (ABTS®=2,2'-azino-di-[3-ethyl-benzthiazoline sulphonic acid(6)]-diammonium salt) and the absorbance was measured after 30 minutes at 405 nm as a measure for the analyte concentration. This value was compared with the absorbance that was obtained by incubation with the monoclonal antibody ECACC 89112804 alone. If this shows a competition of at least 50% when the antibody to be evaluated is in a 10⁵ -fold excess over the monoclonal antibody ECACC 89112804-enzyme conjugate (250 mU/l) then an epitope overlap is present.

EXAMPLE 5 a) Labelling of antibodies with I-125

The antibodies described in the Examples 1 to 4 or fragments thereof were iodinated with 1 mCi I-125 according to the chloramine-T-method (Biochem.J. 89, 114-123 (1963)). Subsequently the non-reacted iodine is separated on a Sephadex G-50 column (Pharmacia) and the immunoreactivity of the monoclonal antibody was checked in an ELISA.

b) Localization of tumours with monoclonal antibody labelled with iodine

Naked mice (Balb/c nu/nu) are inoculated subcutaneously with 3-5×10¹⁰ HeLa cells (type 2). After ca. 8 weeks those mice which had formed a solid tumour were injected with 200 μl KI (potassium iodide) (1 mg/ml) in PBS (phosphate-buffered saline). 24 h after the KI injection ca. 50 μg of the labelled monoclonal antibody or 70 μg of the corresponding antibody fragment are injected. The distribution of the radioactivity is observed over 20 days with the aid of an autoradiographic instrument (e.g. from General Electric). An accumulation of the radioactivity can be observed in the region of the solid tumour as a result of the specific binding to both monoclonal antibodies.

Details of the deposit of the two aforementioned cell lines are shown in the following.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 2                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1394 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 33..1232                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        CGGGGGTTGCTCCGTCCGTGCTCCGCCTCGCCATGACTTCCTACAGCTATCGC53                        MetThrSerTyrSerTyrArg                                                           15                                                                            CAGTCGTCGGCCACGTCGTCCTTCGGAGGCCTGGGCGGCGGCTCCGTG101                            GlnSerSerAlaThrSerSerPheGlyGlyLeuGlyGlyGlySerVal                               10 1520                                                                        CGTTTTGGGCCGGGGGTGGCTTTTCGCGCGCCCAGCATTCACGGGGGC149                            ArgPheGlyProGlyValAlaPheArgAlaProSerIleHisGlyGly                               2530 35                                                                        TCCGGCGGCCGCGGCGTATCCGTGTCCTCCGCCCGCTTTGTGTCCTCG197                            SerGlyGlyArgGlyValSerValSerSerAlaArgPheValSerSer                               404550 55                                                                      TCCTCCTCGGGGGGCTACGGCGGCGGCTACGGCGGCGTCCTGACCGCG245                            SerSerSerGlyGlyTyrGlyGlyGlyTyrGlyGlyValLeuThrAla                               606 570                                                                        TCCGACGGGCTGCTGGCGGGCAACGAGAAGCTAACCATGCAGAACCTC293                            SerAspGlyLeuLeuAlaGlyAsnGluLysLeuThrMetGlnAsnLeu                               7580 85                                                                        AACGACCGCCTGGCCTCCTACCTGGACAAGGTGCGCGCCCTGGAGGCG341                            AsnAspArgLeuAlaSerTyrLeuAspLysValArgAlaLeuGluAla                               9095 100                                                                       GCCAACGGCGAGCTAGAGGTGAAGATCCGCGACTGGTACCAGAAGCAG389                            AlaAsnGlyGluLeuGluValLysIleArgAspTrpTyrGlnLysGln                               105110115                                                                      GGGCCTGGGCCCTCCCGCGACTACAGCCACTACTACACGACCATCCAG437                            GlyProGlyProSerArgAspTyrSerHisTyrTyrThrThrIleGln                               120125130 135                                                                  GACCTGCGGGACAAGATTCTTGGTGCCACCATTGAGAACTCCAGGATT485                            AspLeuArgAspLysIleLeuGlyAlaThrIleGluAsnSerArgIle                               140145 150                                                                     GTCCTGCAGATCGACAACGCCCGTCTGGCTGCAGATGACTTCCGAACC533                            ValLeuGlnIleAspAsnAlaArgLeuAlaAlaAspAspPheArgThr                               155160 165                                                                     AAGTTTGAGACGGAACAGGCTCTGCGCATGAGCGTGGAGGCCGACATC581                            LysPheGluThrGluGlnAlaLeuArgMetSerValGluAlaAspIle                               170175180                                                                      A ACGGCCTGCGCAGGGTGCTGGATGAGCTGACCCTGGCCAGGACCGAC629                           AsnGlyLeuArgArgValLeuAspGluLeuThrLeuAlaArgThrAsp                               185190195                                                                      CTGGAGATG CAGATCGAAGGCCTCAAGGAAGAGCTGGCCTACCTGAAG677                           LeuGluMetGlnIleGluGlyLeuLysGluGluLeuAlaTyrLeuLys                               200205210215                                                                   AAGAAC CATGAGGAGGAAATCAGTACGCTGAGGGGCCAAGTGGGAGGC725                           LysAsnHisGluGluGluIleSerThrLeuArgGlyGlnValGlyGly                               220225230                                                                      CAGGT GAGTGTGGAGGTGGATTCCGCTCCGGGCACCGATCTCGCCAAG773                           GlnValSerValGluValAspSerAlaProGlyThrAspLeuAlaLys                               235240245                                                                      ATCCTGA GTGACATGCGAAGCCAATATGAGGTGATGGCCGAGCAGAAC821                           IleLeuSerAspMetArgSerGlnTyrGluValMetAlaGluGlnAsn                               250255260                                                                      CGGAAGGATGCT GAAGCCTGGTTCACCAGCCGGACTGAAGAATTGAAC869                           ArgLysAspAlaGluAlaTrpPheThrSerArgThrGluGluLeuAsn                               265270275                                                                      CGGGAGGTCGCTGGCCACACG GAGCAGCTCCAGATGAGCAGGTCCGAG917                           ArgGluValAlaGlyHisThrGluGlnLeuGlnMetSerArgSerGlu                               280285290295                                                                   GTTACTGACCTGCGGCG CACCCTTCAGGGTCTTGAGATTGAGCTGCAG965                           ValThrAspLeuArgArgThrLeuGlnGlyLeuGluIleGluLeuGln                               300305310                                                                      TCACAGCTGAGCATGA AAGCTGCCTTGGAAGACACACTGGCAGAAACG1013                          SerGlnLeuSerMetLysAlaAlaLeuGluAspThrLeuAlaGluThr                               315320325                                                                      GAGGCGCGCTTTGGAGCC CAGCTGGCGCATATCCAGGCGCTGATCAGC1061                          GluAlaArgPheGlyAlaGlnLeuAlaHisIleGlnAlaLeuIleSer                               330335340                                                                      GGTATTGAAGCCCAGCTGGCGGAT GTGCGAGCTGATAGTGAGCGGCAG1109                          GlyIleGluAlaGlnLeuAlaAspValArgAlaAspSerGluArgGln                               345350355                                                                      AATCAGGAGTACCAGCGGCTCATGGACATCAA GTCGCGGCTGGAGCAG1157                          AsnGlnGluTyrGlnArgLeuMetAspIleLysSerArgLeuGluGln                               360365370375                                                                   GAGATTGCCACCTACCGCAGCCTGCTCG AGGGACAGGAAGATCACTAC1205                          GluIleAlaThrTyrArgSerLeuLeuGluGlyGlnGluAspHisTyr                               380385390                                                                      AACAATTTGTCTGCCTCCAAGGTCCTC TGAGGCAGCAGGCTCTGGGG1252                           AsnAsnLeuSerAlaSerLysValLeu                                                    395400                                                                         CTTCTGCTGTCCTTTGGAGGGTGTCTTCTGGGTAGAGGGATGGGAAGGAAGGGACCCTTA1312               CCCCCGGCTCT TCTCCTGACCTGCCAATAAAAATTTATGGTCCAAGGGAAAAAAAAAAAA1372              AAAAAAAAAAAAAAAAAAAAAA1394                                                     (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 400 amino acids                                                    (B) TYPE: amino acid                                                            (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetThrSerTyrSerTyrArgGlnSerSerAlaThrSerSerPheGly                               151015                                                                         GlyLeuGlyGlyGlySerVa lArgPheGlyProGlyValAlaPheArg                              202530                                                                         AlaProSerIleHisGlyGlySerGlyGlyArgGlyValSerValSer                               3540 45                                                                        SerAlaArgPheValSerSerSerSerSerGlyGlyTyrGlyGlyGly                               505560                                                                         TyrGlyGlyValLeuThrAlaSerAspGlyLeuLeuAlaGlyAsnGlu                               65 707580                                                                      LysLeuThrMetGlnAsnLeuAsnAspArgLeuAlaSerTyrLeuAsp                               859095                                                                         LysValArg AlaLeuGluAlaAlaAsnGlyGluLeuGluValLysIle                              100105110                                                                      ArgAspTrpTyrGlnLysGlnGlyProGlyProSerArgAspTyrSer                               115 120125                                                                     HisTyrTyrThrThrIleGlnAspLeuArgAspLysIleLeuGlyAla                               130135140                                                                      ThrIleGluAsnSerArgIleValLeuGlnIleAspAsnAl aArgLeu                              145150155160                                                                   AlaAlaAspAspPheArgThrLysPheGluThrGluGlnAlaLeuArg                               16517017 5                                                                     MetSerValGluAlaAspIleAsnGlyLeuArgArgValLeuAspGlu                               180185190                                                                      LeuThrLeuAlaArgThrAspLeuGluMetGlnIleGluGlyLeuLys                                195200205                                                                     GluGluLeuAlaTyrLeuLysLysAsnHisGluGluGluIleSerThr                               210215220                                                                      LeuArgGlyGlnValGlyGlyGlnValSer ValGluValAspSerAla                              225230235240                                                                   ProGlyThrAspLeuAlaLysIleLeuSerAspMetArgSerGlnTyr                               245250 255                                                                     GluLeuMetAlaGluGlnAsnArgLysAspAlaGluAlaTrpPheThr                               260265270                                                                      SerArgThrGluGluLeuAsnArgGluValAlaGlyHisThrGl uGln                              275280285                                                                      LeuGlnMetSerArgSerGluValThrAspLeuArgArgThrLeuGln                               290295300                                                                      GlyLeuGluIleGluLeu GlnSerGlnLeuSerMetLysAlaAlaLeu                              305310315320                                                                   GluAspThrLeuAlaGluThrGluAlaArgPheGlyAlaGlnLeuAla                               325 330335                                                                     HisIleGlnAlaLeuIleSerGlyIleGluAlaGlnLeuAlaAspVal                               340345350                                                                      ArgAlaAspSerGluArgGlnAsnGlnGluTyr GlnArgLeuMetAsp                              355360365                                                                      IleLysSerArgLeuGluGlnGluIleAlaThrTyrArgSerLeuLeu                               370375380                                                                      GluGlyG lnGluAspHisTyrAsnAsnLeuSerAlaSerLysValLeu                              385390395400                                                               

We claim:
 1. Reagent to aid in the detection of malignant diseases, comprising at least two receptors R1 and R2, wherein one of the receptors contains a monoclonal antibody which specifically binds to the sequence 311 to 335 of cytokeratin 19 and other receptor contains a monoclonal antibody which specifically binds to the sequence 346 to 359 of cytokeratin
 19. 2. Reagent according to claim 1, wherein receptor R1 is a biotin conjugate of a monoclonal antibody which specifically binds to amino acid sequence 311 or 346 to 359 of cytokeratin 19 and can be bound to a solid phase which is coated with streptavidin.
 3. Reagent according to claim 2, wherein said reagent contains the antibody which specifically binds to the sequence 311 to 335 of cytokeratin 19 produced by the cell line ECACC 89112803 or is an antibody which binds to the same epitope as the antibody produced by the cell line ECACC
 89112803. 4. Reagent according to claim 2, wherein said reagent contains the antibody which specifically binds to the sequence 346 to 359 of cytokeratin 19 produced by the cell line ECACC 89112804 or is an antibody which binds to the same epitope as the antibody produced by the cell line ECACC
 89112804. 5. The monoclonal antibody produced by the cell line ECACC
 89112803. 6. The monoclonal antibody produced by the cell line ECACC
 89112804. 7. Cell line ECACC
 89112803. 8. Cell line ECACC
 89112804. 9. Method for screening for malignant diseases, comprising the steps of:incubating a sample of a body fluid with at least two receptors R1 and R2 in a liquid phase, wherein one of the two receptors contains a monoclonal antibody which specifically binds to the amino acid sequence 311 to 335 of cytokeratin 19 and the other receptor contains a monoclonal antibody which specifically binds to the amino acid sequence 346 to 359 of cytokeratin 19, to produce a signal change by binding of the receptors R1 and R2 to the substance to be detected in the sample solution, and detecting the signal change in the sample caused by the binding as an indication of the presence of a malignant disease.
 10. The method according to claim 9, wherein the antibody specifically binding to the sequence 311 to 335 is produced by the cell line ECACC 89112803 or is an antibody which specifically binds to the same epitope as the antibody produced by the line ECACC
 89112803. 11. The method according to claim 9, wherein the antibody specifically binding to the sequence 346 to 359 is produced by the cell line ECACC 89112804 or is an antibody which specifically binds to the same epitope as the antibody produced by the cell line ECACC
 89112804. 12. Method according to claim 9, wherein R1 is bound to a solid phase and R2 is detectably labelled.
 13. The method according to claim 12 further comprising separating said solid phase from said liquid phase and detecting R2 in one of the two phases.
 14. The method according to claim 13, wherein said solid phase is coated with streptavidin and receptor R1 is a biotin conjugate of one of said two antibodies, and the binding of R1 to the solid phase is effected by the binding of biotin to streptavidin.
 15. The method according to claim 12, wherein R2 is labelled with a radioactive isotope, with an enzyme, or with a substance which generates fluorescent or NMR signals. 